Radiological feature heterogeneity supports etiological diversity among patient groups in Meniere’s disease

We aimed to determine the prevalence of radiological temporal bone features that in previous studies showed only a weak or an inconsistent association with the clinical diagnosis of Meniere’s disease (MD), in two groups of MD patients (n = 71) with previously established distinct endolymphatic sac pathologies; i.e. the group MD-dg (ES degeneration) and the group MD-hp (ES hypoplasia). Delayed gadolinium-enhanced MRI and high-resolution CT data were used to determine and compare between and within (affected vs. non-affected side) groups geometric temporal bone features (lengths, widths, contours), air cell tract volume, height of the jugular bulb, sigmoid sinus width, and MRI signal intensity alterations of the ES. Temporal bone features with significant intergroup differences were the retrolabyrinthine bone thickness (1.04 ± 0.69 mm, MD-hp; 3.1 ± 1.9 mm, MD-dg; p < 0.0001); posterior contour tortuosity (mean arch-to-chord ratio 1.019 ± 0.013, MD-hp; 1.096 ± 0.038, MD-dg; p < 0.0001); and the pneumatized volume (1.37 [0.86] cm3, MD-hp; 5.25 [3.45] cm3, MD-dg; p = 0.03). Features with differences between the affected and non-affected sides within the MD-dg group were the sigmoid sinus width (6.5 ± 1.7 mm, affected; 7.6 ± 2.1 mm, non-affected; p = 0.04) and the MRI signal intensity of the endolymphatic sac (median signal intensity, affected vs. unaffected side, 0.59 [IQR 0.31–0.89]). Radiological temporal bone features known to be only weakly or inconsistently associated with the clinical diagnosis MD, are highly prevalent in either of two MD patient groups. These results support the existence of diverse—developmental and degenerative—disease etiologies manifesting with distinct radiological temporal bone abnormalities.

Meniere's disease (MD) is believed to be an inner ear disorder with diverse, yet unknown, etiologies [1][2][3] . Although a plethora of temporal bone (TB) abnormalities has been described in MD patients, including, amongst others, poor pneumatization and arrested growth of several bone features, as well as vascular anomalies of the sigmoid sinus (Ss) and the jugular bulb (JB), these features were either highly variably expressed between individual patients or inconsistently prevalent among patient cohorts [4][5][6][7][8] ; thus, drawing their pathogenic significance into question. Recently emerging genotypes [9][10][11] , endotypes 12,13 , and clinical phenotypes 14,15 in MD begin to delineate more homogenous patient groups, eventually opening up new opportunities to investigate the long-suspected diverse disease etiologies. 'Endotypes' refers to disease subtypes defined by specific biological mechanisms that lead to the same clinical manifestation 16 . Considering these ongoing advances, it appears it is worth revisiting some of the previously reported TB features under the new hypothesis that each feature is associated with a distinct disease etiology and thus, is prevalent only in a subset of MD patients. Using CT and MRI data, we divided a retrospective cohort of MD patients into two previously defined groups with suspected different disease etiologies, as indicated by their different histopathologies of the endolymphatic sac (ES) 12 and the distinguishing radiological (CT/MRI) surrogate markers for both ES pathologies 13 . Accordingly, patients either (1) had developmental hypoplasia of the ES as indicated by the radiological surrogate marker-the angular trajectory of the bony vestibular aqueduct being ≥ 140°-and were assigned to the "MD-hp group", or (2) had progressive degeneration of the ES as indicated by the radiological angular trajectory of the bony vestibular aqueduct being ≤ 120°, and were assigned to the "MD-dg group" 12,13,15 . In this study we then compared between groups as well as between clinically affected and non-affected TBs within groups, radiological TB features, including geometrical features (contours and dimensions), the volume of the air cell tracts, the height of the jugular bulb, the width of the sigmoid sinus, and Gd-MR signal intensity alterations of the ES. Some of the geometrical features were cross-examined in histologically processed temporal bones from deceased MD-hp and MD-dg patients. We aimed to identify radiological features that would further support the existence of etiologically distinct patient groups, and which could be used as additional clinical markers to distinguish MD-dg and MD-hp patients.

Materials and methods
Study population. We included a retrospective cohort (n = 71) of patients with definite uni-or bilateral MD 17 , for whom high-resolution CT (HRCT) and/or gadolinium-enhanced MRI (Gd-MRI) data including a delayed Gd-enhanced 3D-IR sequence of the TBs were available. This cohort was already described in another recent retrospective study 15 . The cohort characteristics are presented in Table 1.
Patient group assignment. Patients were assigned either to the MD-hp or the MD-dg group according to a previously described method in which the angular trajectory of the vestibular aqueduct (ATVA) in the axial imaging plane serves as an indicator for ES hypoplasia (angle ≥ 140°) and ES degeneration (angle ≤ 120°), respectively 13 . Patients with Meniere's syndrome secondary to other disease processes 18 were not included in this cohort.
Archival human TB specimens. To investigate the true (histological) thickness of the bony covering of the posterior semicircular canal, 33 specimens from cases with a clinical diagnosis of definite MD and 30 specimens from cases with normal age-related audiometric threshold patterns and no history of otologic disease, all processed for light microscopic studies 18 , were used in this study. Among the specimens from deceased MD patients, the ES was histologically determined as either hypoplastic, and the individual specimen was assigned to the MD-hp subgroup (n = 15), or as degenerated, and the individual specimen was assigned to the MD-dg subgroup (n = 18) 12 .
Clinical imaging. All patients underwent 3 T MRI using a 64-channel head coil of the TBs to rule out intraor retrocochlear pathologies and to detect endolymphatic hydrops. Using previously published protocols, 4 h after intravenous contrast administration (Gadovist; Bayer-Schering Pharma, Berlin, Germany; 1.0 mmol/mL at a dose of 0.2 mmol/kg), a 3D real inversion recovery sequence was acquired using the following parameters: field of view of 190 mm, section thickness of 0. Dedicated TB HRCT imaging was available for a subset of patients (n = 12). HRCT data were reconstructed separately for each TB in the axial plane using a standard bone algorithm at 0.6 mm. Imaging analysis was www.nature.com/scientificreports/ performed as described in the following paragraphs by investigators blinded to any clinical information or radiological diagnosis. All distance and length measurements were performed using ImageJ/Fiji software (version 1.52q 21 ). 3D volumetric measurements of TB pneumatization were performed using the 3D Slicer software (version 4.10.2 22 ).

Anteroposterior and mediolateral TB dimensions. Length and width measurements were performed
on Gd-MRI data (axial T2-weighted sequences; field of view of 220 mm, section thickness of 2 mm, matrix size 384 × 384). From the cochlear apex (measurement point 'Co') and the center of the horizontal semicircular canal (vestibular; measurement point 'Ve'), the distances to the posterior (ΔCo-post, ΔVe-post), medial (ΔCo-med, ΔVe-med) and lateral (ΔCo-lat, ΔVe-lat) TB surfaces were determined (Fig. 1a). Moreover, the distance between the most posterior border of the posterior semicircular canal (pSCC) and the posterior TB surface (ΔpSCCpost) was determined (Fig. 1a′) in the radiological data, as well as in histologically processed TB specimens.
Tortuosity of the posterior TB surface. The tortuosity of the posterior TB surface was analyzed in the axial plane at the level of the operculum (opening of the vestibular aqueduct into the posterior cranial fossa). A line segment of the posterior TB border with its length defined by a circle with its center at the operculum and a radius of 2 cm was extracted manually in Fiji 21 using the "Export_multiset" macro 23 (Fig. 1a). The tortuosity T was defined as the arc-chord ratio T = L/D, where L is the actual length of the line segment, and D is the straightline distance between the endpoints of line segment. Tortuosity has a value of 1 for a straight line and > 1 for twisted lines. All numerical computations were performed in Python (Python Language Reference, version 3.6, Python Software Foundation, Wilmington, DE, USA).

Volumetric quantification of TB pneumatization.
In patients in whom dedicated HRCT imaging of the TBs was available, we quantified the volume of the TB air cell tracts, excluding the middle ear space and pneumatized spaces in the petrous apex, by manually segmenting the pneumatized areas on axial sections with a threshold range of − 1024 to 200 Hounsfield units (HU) for pneumatized areas and 600-3052 HU for bone ( Fig. 1b). The respective volumes were reconstructed and calculated using the segmentation module and the segment statistics module in the 3D Slicer software.

Sigmoid sinus (Ss) width and jugular bulb (JB) height.
The width of the right and left Ss was measured along horizontal lines at the level of the most lateral extension of the cerebellar hemispheres in a coronal T2-weighted MR image (Fig. 1c). The distance between the most cranial extension of the JB and the floor of the internal auditory canal (IAC) was measured along a vertical line in a coronal T2-weighted MR image (Fig. 1d).

Inversion recovery signal intensity of the ES within the vestibular aqueduct in Gd-MRI scans.
In Gd-MRI data sets (3D inversion recovery sequence acquired 4 h post i.v. Gd injection) from MD-dg patients with unilateral MD (n = 40), we manually segmented the MR signal along the vestibular aqueduct from its proximal end near the vestibulum, corresponding to the location of the endolymphatic duct within the vestibular aqueduct, to its posterior end distal to the operculum, corresponding to the location of the extraosseous ES. Segmentations were performed in both TBs from each patient using the segment editor module in the 3D Slicer software (Fig. 1e). For each vestibular aqueduct, the mean signal intensity (mSI; brightness value in arbitrary units [a.u.]) was determined and normalized to the SI of the cerebrospinal fluid (CSF) signal in the IAC on the clinically non-affected side (normalized mSI [nmSI]). The ratio between the nmSI of the affected (nmSI affected ) and the non-affected (nmSI non-affected ) sides was determined (nmSI affected /nmSI non-affected ).
Statistical analysis. All statistical tests were selected before data collection. Each ear was analyzed as a separate experimental unit. Although the two ears of a same are never completely independent, treating ears as separate experimental units is justified by the fact that the two ears rarely match in their predisposition and clinical presentation in MD. To compare measurements among > 2 groups, one-way ANOVA was used. Following one-way ANOVA, pairs of means were compared, and the Holm-Sidak test was used to correct for multiple comparisons.

Results
A total of 71 MD patients were included (one patient from the cohort was excluded due to missing imaging data that were required for the present analyses). Of those, 55 patients (77.5%) had previously been assigned to the MD-dg subgroup, and 16 patients (22.5%) had previously been assigned to the MD-hp subgroup 15 (Table 1). www.nature.com/scientificreports/   Fig. 2b); however, no specimen exhibited true dehiscence of the pSCC. Exemplary MR images of all TB geometrical features are shown in an MD-hp patient (Fig. 2i) and in an MD-dg patient (Fig. 2j).
Intergroup comparison of the two-dimensional shape of the posterior TB surface (Fig. 1a) demonstrated a significantly lower line curvature (arch-to-chord ratio), i.e., a flatter posterior TB surface in the MD-hp group than in the MD-dg group (MD-dg: 1.096 [0.038]; MD-hp: 1.019 [0.013]; p < 0.0001; Fig. 3). Exemplary outlines of the posterior TB surface in an MD-dg and an MD-hp patient are depicted in Fig. 2i-j (yellow broken lines).

Hypoplastic mastoid air cell tracts in the MD-hp group.
A total of 23 TB HRCT data sets from 12 patients were included in the analysis. One TB HRCT data set was excluded due to an anterior mastoidectomy that was performed for cochlear implantation. Although carried out in a subsample of the present study, an intergroup comparison of the total pneumatization volume (Fig. 1b)

Reduced Ss width and JB height in affected ears in the MD-dg group. Intergroup comparison of
the Ss width, as measured at the level of the most lateral extension of the cerebellar hemispheres in the coronal imaging plane (Fig. 1c), demonstrated no significant difference between groups (MD-dg: 6.45 [1.74] mm; MD-hp 6.41 [2.29] mm; p = 0.94; Fig. 5a). Within the MD-dg group, the Ss width on the clinically affected side was significantly smaller than that on the non-affected side (affected side: 6.5 [1.7] mm; non-affected side: 7.6 [2.1] mm; p = 0.04; Fig. 5a). Intergroup comparison of the maximum height of the JB, as measured by the distance between the cranial border of the JB and the floor of the IAC in the coronal imaging plane (Fig. 1d)

Reduced inversion recovery SI of the distal ES in clinically affected ears of the MD-dg group.
The mean nmSI affected /nmSI non-affected ratio from all MD-dg patients with unilateral MD was determined to be 0.56 (interquartile range [IQR] 0.34-0.85). A ratio < 1, indicating a relative decrease in SI on the clinically affected side compared to the non-affected side (Fig. 6), was found in 36 (90%) of MD-dg patients. The most pronounced and consistent decrease in inversion recovery SI was overall observed in the most-posterior TB region near the operculum, which corresponds to the location of the extraosseous ES (red arrows in Fig. 6).

Discussion
For many of the radiological TB features (re)investigated in the present study, inconclusive 6 or contradictory 25,26 prevalence data were reported previously in MD patients. Here, we instead distinguished two etiopathologically distinct patient groups, the MD-hp group (~ 30% of all MD patients), and the MD-dg group (~ 70% of all MD patients), characterized by developmental hypoplasia and degeneration of the ES, respectively 12,15 . We demonstrated several of the examined radiological TB features to be predominantly prevalent in either of the two patient groups. Furthermore, the most TB features appeared to be linked to the respective group's etiopathogenic context-i.e. developmental vs. degenerative.
In the MD-hp group, the pathognomonic histopathology of ES hypoplasia features a lower-than-normal number of ES epithelial cells, and the remaining epithelium features diminished protein expression of ion channels and transporters 12 . Impaired local ion transport in the hypoplastic ES most likely has propagating disrupting effects on ion and fluid homeostasis throughout the inner ear. Thereby, ES hypoplasia presumably critically contributes to the pathogenesis of endolymphatic hydrops and MD 12,27 . The etiology of ES hypoplasia remains unknown. Its morphological resemblance with the fetal/newborn human ES 28 points toward a premature developmental arrest 13,29 . The invariably associated hypoplastic bony vestibular aqueduct, which takes an abnormally straight and short course through the posterior TB portion (see material and methods section, diagnostic criteria for the MD-hp group 13 ), further indicates a developmental TB pathology. Here, we identified additional hypoplastic TB features, including poorly pneumatized air cell tracts and a thinned, abnormally flat posterior TB surface, in the MD-hp group. The combined presence of all hypoplastic features in the retrolabyrinthine bone suggests a regional developmental defect, possibly due to a distinct genetic etiology that remains to be investigated. We suppose that the hypoplastic bone features are merely epiphenomena, whereas ES hypoplasia (loss of ES function) is www.nature.com/scientificreports/  , did not demonstrate any signs of "true" pSCC dehiscence. We here found a ΔpSCC-post < 2 mm to be exclusive to the MD-hp group, meaning a value > 2 mm virtually excluded the MD-hp endotype and may thus be used as an additional radiological marker in radiological endotyping, in addition to the angular trajectory of the vestibular aqueduct 13 .
In the MD-dg group, the pathognomonic histopathology of ES degeneration features deteriorating ES epithelial cells and local fibrotic remodeling. The resulting loss of epithelial ion channels and transporters 12 probably www.nature.com/scientificreports/ has the same pathophysiological impact on inner ear fluid homeostasis in MD as presumed for ES hypoplasia (see previous paragraph 12,27 ). The underlying cause of ES degeneration is unknown. One possible pathomechanism that could primarily and selectively damage the ES epithelium is ischemic injury. The microvascular supply of the ES is terminal and is largely separate from that of the cochlea and the vestibular labyrinth (no or few anastomoses) [31][32][33] , and thus likely predisposes for local ischemic events. Hypotheses on a vascular compromise   www.nature.com/scientificreports/ to the inner ear as a causal factor in MD were previously considered. Radiology studies investigated vascular anomalies in the TB region and compared the course and caliber of major blood vessels between MD patients and control cohorts [34][35][36] ; mostly though with variable/ambiguous/inconclusive data among studies. Here, we re-assessed radiological parameters (course and caliber, respectively) of the Ss and the JB, both of which directly receive blood from inner ear venules 37 and are thus part of the inner ear venous drainage system. We found a significantly decreased cross-sectional diameter (smaller caliber) of the Ss and a significantly increased distance between the JB and the IAC (divergent course) in clinically affected TBs as compared to non-affected TBs in the MD-dg group. However, both features were highly variably expressed with no clear-cut distinction (standard deviation errors overlapping between groups) between clinically affected and non-affected TBs from MD-dg. Whether a smaller caliber of the Ss or the divergent course of the JB affect the venous drainage from the inner ear in patterns that cause selective ischemia and degeneration of the ES is not known. Notably, side asymmetries in the caliber and course of these venous vessels are well known and widespread norm variants 38 . Together, evidence for the presence of vascular anomalies in major vessels of the inner ear's venous drainage system in the MD-dg group is inconclusive. Other pathomechanisms, such as e. g. local inflammatory injury 39,40 , may-at least in some members of the MD-dg group-better explain the degenerative histopathology of the ES. Upon further analysis, we almost invariably detected a relative decrease in the inversion recovery Gd-MRI signal that originated from the ES in the clinically affected inner ears of MD-dg patients. The focal SI loss was most pronounced in the distal (extraosseous) ES, which is also the ES portion that is most severely affected by epithelial degeneration 12 . Thus, the inversion recovery Gd-MRI signal loss may be a radiological correlate of this histopathology. In contrast, in four MD-dg patients, we found a 1.5-fold or higher inversion recovery Gd-MRI signal from the ES on the clinically affected side compared to that on the non-affected side. Focal signal hyperintensity of the ES upon MRI-Gd imaging was already associated with a broad range of cochleovestibular symptoms (not with MD) and was interpreted as active inflammation of the ES ("endolymphatic sacitis" 40 ). For the present four cases, however, the available clinical records did not indicate an early disease stage and did not mention any acute disease activity at the time of MRI data acquisition, which impedes any interpretation of their disparate MRI findings.

Conclusion
Expressional variability of radiological TB features among MD patients can be explained by the existence of, at least two, different disease endotypes. Future studies investigating the etiologies of MD, instead of including patients solely based on the clinical diagnosis of MD, may consider patient (sub)groups with genetically, pathologically, or clinically distinct endotypes to gather more meaningful data.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.